Immunoassay methods for the detection of HIV-1 antibodies employing envelope muteins containing hypervariable domain deletions

ABSTRACT

HIV-1 envelope muteins are provided comprising deletions within the hypervariable domains of the poly-peptides. Methods of using these proteins in immunoassay and to elicit antibody production are also disclosed, as well as materials and methods useful for producing the muteins by recombinant DNA technology.

This application is a continuation of application Ser. No. 08/006,252filed on 19 Jan. 1993, now abandoned, which is a continuation ofapplication Ser. No. 07/243,944 filed 13 Sep. 1988, now abandoned.

TECHNICAL FIELD

The present invention is directed to novel analogs (muteins) of theenvelope proteins from human immunodeficiency virus type 1 (HIV-1),methods of making the analogs, DNA sequences encoding the analogs, andmethods of using the analogs, for example, in immunoassays and vaccinecompositions.

BACKGROUND

HIV-1, and a recently identified related virus named HIV-2, are theknown causative agents of Acquired Immune Deficiency Syndrome (AIDS).Many isolates of HIV-1 have been identified and sequenced. A strikingfeature of these independent isolates is substantial genomic and aminoacid variation, centered particularly in the envelope gene and proteins.This variation among HIV-1 isolates has important implications for bothAIDS diagnostics and potential vaccines.

Starcich et al., (1986) Cell 45:637-648, reports on the geneticvariation in five independent HIV-1 isolates, as well as variations indeduced amino acid sequences. Both conserved and variable regions wereobserved.

Coffin, (1986) Cell 46:1-4, is a review directed to the variation in theenvelope of HIV-1, and the possible mechanism which brings about thevariation. Coffin hypothesized that the highly variable domains, termed"hypervariable" domains, are masking epitopes within the conserveddomains from being available for neutralizing antibodies orcell-mediated immune responses. It is concluded that vaccinationstrategy for HIV-1 should be directed towards developing the ability toprovoke an immune response directed against the conserved regions of theenvelope despite the presence of masking variable domains.

Modrow et al., (1987) J. Virol. 61:570-578, is also directed tocomparison of the amino acid sequences of various HIV-1 isolates.Computer analysis was employed to predict epitopes in the envelopeprotein, and it was found that the majority of predicted epitopes werelocated in the hypervariable regions. See, e.g., FIG. 1 and Tables 1 &2, incorporated herein by reference.

Hahn et al., (1986) Science 232:1548-1553, discloses the sequencevariations in a series of HIV-1 isolates from a single individual,particularly in the envelope. See also Saag et al., (1988) Nature334:440-444; Fisher et al., (1988) Nature 334:444-447.

Rusche et al., (1988) Proc. Natl. Acad. Sci. USA 85:3198-3202, disclosesthat a short peptide having the sequence of an HIV-1 isolate in thethird hypervariable domain of the envelope protein was able to absorbisolate-specific neutralizing antibodies from antisera, and that anothergroup was able to elicit isolate-specific neutralizing antibodies byimmunization with a peptide from the same domain. It is suggested thatsince this neutralizing epitope is found in one of the hypervariabledomains that a possible vaccination strategy is to prepare a subunitantigen made up of the critical epitope from multiple isolates. See alsoLooney et al. (1988) Science 241:357-359.

Goudsmit et al., (1988) Proc. Natl. Acad. Sci. USA 85:4478-4482, reportson the neutralizing ability of antibodies raised in chimpanzees infectedwith various HIV-1 isolates, and a rabbit immunized with an envelopesubunit. The epitope identified by Rusche et al. is reported to be anisolate-specific neutralizing epitope, and that it is immunodominant inHIV-1 chimpanzees.

Due in large part to the generally accepted view that attenuated orkilled virus vaccines for AIDS are not feasible, the primary focus forthe development of vaccines has been the subunit antigens. Severalgroups have suggested that short oligopeptides from the envelope domainwould make suitable subunit antigens. Other groups are pursuing liverecombinant virus vaccines, natural or recombinant viral polypeptidevaccines, or anti-idiotype vaccines. See, e.g., Koff et al., (1988)Science 241:426-432 (and references cited therein).

A continuing need exists to develop new and better HIV-1 envelopeanalogs, as well as polypeptides that avoid isolate-specific immuneinteractions for use in diagnostics and as potential vaccines.

SUMMARY OF THE INVENTION

The present invention is directed to HIV-1 envelope analogs (muteins)comprising the constant domains of gp120env or gp160env, but lacking atleast one epitope from a hypervariable domain. Despite the reports inthe literature suggesting the importance of the epitopes in thehypervariable domains (e.g., immunodominant and/or neutralizing), it hassurprisingly been discovered that the muteins of the present inventionare useful as diagnostic reagents exhibiting at least as great orgreater reactivity to antibodies raised against diverse isolates, and asantigens in raising nonisolate-specific antibodies upon immunization ofa mammal.

In one embodiment, the present invention comprises an improved analog ofHIV-1 gp120env or gp160env wherein the improvement comprises thedeletion of at least one epitope within a hypervariable domain, whileretaining the domains conserved among HIV-1 isolates.

In another embodiment, the present invention is directed to apolypeptide comprising epitopes bound by antibodies to HIV-1 gp120env orgp160env and an amino acid sequence according to the formula:

    C1-V1-V2-C2-V3-C3-V4-C4-V5-C5

wherein:

C1 is an amino acid sequence substantially homologous to Ser29 throughCys130 of HIV-1 SF2;

C2 is an amino acid sequence substantially homologous to Cys 199 throughLeu291 of HIV-1 SF2;

C3 is an amino acid sequence substantially homologous to Ser366 throughCys387 of HIV-1 SF2;

C4 is an amino acid sequence substantially homologous to Cys415 throughGly456 of HIV-1 SF2;

C5 comprises an amino acid sequence substantially homologous to Phe466through Arg509 or Leu855 of HIV-1 SF2;

V1 is an amino acid sequence of 0 to a maximum of about 30 residues;

V2 is an amino acid sequence of 0 to a maximum of about 50 residues;

V3 is an amino acid sequence of 0 to a maximum of about 90 residues;

V4 is an amino acid sequence of 0 to a maximum of about 30 residues; and

V5 is an amino acid sequence of 0 to a maximum of about 10 residues;

with the proviso that at least one of the V domains selected from thegroup consisting of V1, V2, V3, V4 and V5 contains no more than aboutone-third of the said maximum number of residues for the V domain.

Still another embodiment of the present invention is directed to apolypeptide analog of HIV-1 gp120env or gp160env comprising (a) about300 to about 850 amino acid residues in length; (b) constant domains, inthe N-terminal to the C-terminal direction, Ser29-Cys130 of HIV-1 SF2(C1), Cys199-Leu291 of HIV-1 SF2 (C2), Ser366-Cys387 (C3), Cys415-Gly456(C4) of HIV-1 SF2, and Phe466-Arg509 or Phe466-Leu855 of HIV-1 SF2 (C5),or domains substantially homologous to said C1, C2, C3, C4 or C5; and(c) the intervening domains, if any, located between said constantdomains comprising sequences found between substantially homologousconstant domains in native HIV-1 gp120env, with the proviso that atleast one of said intervening domains between said constant domains iseither missing or missing an epitope.

The present invention is also directed to an immunoassay for thedetection of antibodies to HIV-1 comprising: (a) providing a liquidsample to be tested for the presence of anti-HIV-1 antibodies; (b)contacting said sample with a polypeptide as described above underconditions whereby any anti-HIV-1 antibodies present in said sample maybind to an epitope to provide sample-contacted polypeptide; and (c)detecting any antibody bound to said sample-contacted polypeptide.

In yet another embodiment, the present invention is directed to a methodof selectively raising antibodies in a mammal to epitopes in theconstant domains of human immunodeficiency virus type 1 (HIV-1) gp120envor gp160env comprising administering to said mammal a polypeptide asdescribed above, whereby antibodies to said polypeptide are produced inresponse to said administration.

The present invention is also directed to a composition useful in suchmethod comprising the polypeptide described above in combination with apharmaceutically acceptable carrier.

The present invention is also directed in another embodiment to a DNAsequence encoding the above polypeptide, as well as a cellular hostcomprising the DNA sequence under the control of transcriptional andtranslational control sequences whereby the polypeptide encoded by theDNA sequence is expressed by the cellular host. The present invention isalso directed to methods of producing the above polypeptide by growing aculture of the above cellular host under conditions whereby thepolypeptide encoded by the above DNA sequence is expressed, andrecovering the polypeptide from the culture.

These and other embodiments of the present invention will be apparent tothose of ordinary skill in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of the wild-type gp120env gene fromHIV-SF2.

FIG. 2 shows the alignment of amino acid sequences for various HIV-1isolates, including SF2, with the constant and variable domainsindicated. Potential N-linked glycosylation sites, for the HXB2 sequenceonly, are indicated by " !"; cysteine residues have "*" above them.

FIG. 3 is a restriction map of mammalian expression vectorpSV7dARV120tpa.

FIG. 4 is a restriction map of mammalian expression vectorpCMV6ARV120tpa.

FIG. 5 is a restriction map of yeast expression vector pHL15.

FIG. 6 contains graphs showing the result of an ELISA testing thereactivity of HIV-1 envelope muteins of the present invention andcontrols against a North American serum panel. The graphs show that forserum samples, the recombinant antigen env SF2-D1-2 is as efficient ormore efficient in detecting sera as is env SF2 wild-type.

FIG. 7 contains graphs showing the result of an ELISA testing thereactivity of HIV-1 envelope muteins of the present invention andcontrols against an African serum panel. The graphs show that for serumsamples, the recombinant antigen env SF2-D1-2 is as efficient or moreefficient in detecting sera as is env SF2 wild-type.

FIG. 8 shows the results of an ELISA using sera from guinea pigsimmunized with envelope muteins according to the present invention orvarious controls.

FIG. 9 shows the results of an ELISA using sera from goats immunizedwith envelope muteins according to the present invention or variouscontrols.

DETAILED DESCRIPTION

HIV-1 is a known virus, of which many isolates have been observed. See,e.g., Levy et al., (1984) Science 225:840; Barre-Sinoussi et al., (1983)Science 220:868; Popovic et al., (1984) Science 224:497; AIDS: Papersfrom Science, 1982-1985 (R. Kulstad ed. 1986); Current Topics in AIDS(M. Gottlieb et al. eds. 1987). The envelope gene of HIV-1 produces aprecursor glycoprotein of approximately 160 mw, referred to as gp160 orgp160env. The precursor is cleaved during expression to provided gp120(gp120env), the major exterior envelope glycoprotein, and gp41(gp41env), the transmembrane protein. Various HIV-1 isolates have beenreported in the literature, including, BH10, PV22, BRU, HXB2, WMJ1,WMJ2, WMJ3, CDC4, HTLV-IIIRF, HTLV-IIIB, Z3, Z6, Z321, MAL, NY5, ELI,JY1, LAV1A, and HAT3. Of particular interest to the present invention isHIV-1 isolate SF2, originally designated ARV2. See, e.g., Levy, U.S.Pat. No. 4,716,102; Levy et al., (1984) Science 225:84; EPO Pub. No.181,150.

The conserved and hypervariable domains of HIV-1 envelope proteins havebeen described previously. See, e.g., Modrow et al., supra. FIG. 1herein is a schematic diagram showing the location of the fivehypervariable regions (V1, V2, V3, V4 and V5) and the five constantregions (C1, C2, C3, C4 and C5) in the envelope gene from HIV-1 SF2.Various deletions encompassing part or all of a hypervariable domain arealso shown (D1, D2, D3, D4 and D5). Two regions of predominantlyhydrophobic amino acids are denoted by dark shading, where thetransmembrane anchor has been labeled TM. The signal sequence ishighlighted at the N-terminus, as well as the processing site separatinggp120env (positions 1-1527) from gp41env (positions 1528-2565).

Hypervariable domains are characterized by a substantial lack ofhomology (e.g., as low as 10%) among independent HIV-1 isolates.Furthermore, there is a substantial variation in length among thehypervariable domains from various isolates due to the prevalence ofinsertion and deletion mutations. Thus, these regions cannot becharacterized from one isolate to the next by having any substantialdegree of amino acid sequence homology, and can only be assigned anapproximate length. The primary characterization of hypervariabledomains is their location within the envelope glycoprotein and theirpresumed tertiary structure (i.e., loops). The conserved or constantdomains, as well as all 18 cysteines in the envelope, are highlyconserved. Corresponding hypervariable domains are found to be locatedidentically relative to surrounding constant domains and cysteines fromone isolate to the next. Furthermore, the tertiary structure of thehypervariable domain appears to be highly conserved; e.g., twononhomologous hypervariable domains from different HIV-1 isolates willusually both exhibit the same three-dimensional conformation, such as anexposed loop. Thus, hypervariable domains from new HIV-1 isolates can bereadily identified by sequencing the new isolates and comparing thesequence to known HIV-1 sequences so that the conserved domains andcysteines are aligned. See, e.g., Modrow et al., supra and FIG. 2herein.

The location of the various domains, both constant and hypervariable,will be described hereinafter with reference to the sequence andnumbering of the HIV-1 SF2 isolate. It is to be understood that this isfor convenience only; the invention is not limited to analogs containingonly HIV-1 SF2 sequences, but also encompasses analogs employingcorresponding domains or sequences from other isolates. A domain fromanother HIV-1 isolate envelope protein can be easily identified ascorresponding to an SF2 sequence by those of ordinary skill in the artby alignment of the conserved domains and the 18 cysteines of bothisolates. Such an alignment is shown in FIG. 2, where the correspondingsequences of 15 HIV-1 isolates are aligned. The cysteine residues aremarked with an asterisk, and potential N-linked glycosylation sites forthe HXB2 isolate are indicated by " !". The cleavage site for the signalpeptide and for the mature gp120env and gp41env proteins are shown,along with the constant domains, C1-C5. Specific deletions, D1-D5, arealso shown in the figure.

The constant domains of the SF2 isolate are as follows: C1,Ser29-Cys130; C2, Cys199-Leu291; C3, Ser366-Cys387; C4, Cys415-Gly456;and C5, which is Phe466-Arg509for gp120env analogs, or Phe466-Leu855 forgp160env analogs. Since these domains are highly conserved among HIV-1isolates, the corresponding sequences from isolates other than SF2 willbe substantially homologous to these SF2 domains; i.e., a minimum ofabout 70-75% amino acid sequence homology, with certain highly conserveddomains exhibiting a minimum of about 80%, or even 85-90% homology. Thedifferences in amino acid sequences found in these conserved domains aregenerally attributable to point mutations in the nucleic acid sequence,as opposed to the deletion and insertion mutations which typify thedifferences in hypervariable domains.

As shown in FIGS. 1 and 2, the hypervariable domains lie between theconserved domains, C1-C5. Not all of the amino acids in theseintervening domains, however, comprise hypervariable regions. Indeed,due to the heterogeneity among HIV isolates in the hypervariable domain,it is not feasible to generally define precise limits of thehypervariable domain. Thus, for the convenience of describing thepresent invention, the domains between the constant domains will bereferred to as "variable" or "intervening" domains, and it will beunderstood that they may comprise both hypervariable regions as well asless variable, yet not highly conserved, sequences. Thus, the analogs ofthe present invention can be described schematically according to thefollowing formula:

    C1-V1-V2-C2-V3-C3-V4-C4-V5-C5                              (I)

C1-C5 and V1-V5 in formula I are defined as described above in theSummary of the Invention. V1-V5 consist of the variable or interveningdomains which contain hypervariable regions. The polypeptides of thepresent invention will contain a deletion in at least one of thesevariable domains when compared to a native gp120env or gp160env. Thedeletions will typically be at least about one-third of the variabledomain, and oftentimes will consist of a deletion of the entire variabledomain. Furthermore, it is also preferred to delete sequences from morethan one of the variable domains, including the deletion of all five ofthe variable domains in their entirety. Thus, in accordance with thepresent invention, any of the variable domains, V1-V5, can be anywherefrom zero to a maximum number of amino acids in length, the maximumbeing an approximation of the longest of such domains found in HIV-1isolates. At least one of the V domains, however, will have deletedtherefrom at least one-third of that maximum number of amino acids, orone-third of the length of the corresponding domain from the homologousHIV-1 isolate.

Since the variable or intervening domains of formula I can comprise morethan the true hypervariable domains, a selected Vn domain can beexpressed by the following formula:

    Sn-HVn-Sn'                                                 (II)

wherein n is 1, 2, 3, 4 or 5 (as in V1, V2, etc.), HVn is ahypervariable domain of x amino acid residues in length, and Sn and Sn'are nonhypervariable sequences flanking HV in the Vn domain, theflanking sequences being y and y' residues in length, respectively. Inthe native protein, the sum of x, y and y' equals the maximum number ofresidues for Vn in a chosen isolate, and y and/or y' may be zero. As forthe analogs of the present invention, the sum of x, y and y' will beanywhere from zero to the maximum number. The actual values for x, y andy' for a particular Vn can be determined by sequence comparison of arepresentative number of HIV-1 isolates. See, e.g., FIG. 2 and Modrow etal., supra.

The following are the intervening domains of the SF2 isolate, which canbe used for comparison purposes to determine the correspondingintervening domains in other HIV-1 isolates. See, e.g., FIG. 2. V1 ofSF2 is about 24 amino acids in length, encompassing Thr131through Asn154and, optionally, Cys155. This C-terminal cysteine is one of the 18highly conserved cysteine residues found in gp120env. The correspondingdomains from independent HIV-1 isolates sequenced to date appears torange from about 22 to about 31 amino acid residues in length. V2 of SF2spans from Ser156 through Ser198. The corresponding domain in otherHIV-1 isolates sequenced to date appears to range from about 39 to about52 amino acid residues in length. V3 of SF2 spans Asn292 through Glu365,and the corresponding domains in other HIV-1 isolates sequenced to dateappears to range from about 88 to about 90 amino acid residues inlength. V4 of SF2 spans from Asn388 through Pro414, and thecorresponding domains from other HIV-1 isolates appear to range fromabout 28 to about 33 residues in length. V5 of SF2 spans from Thr457through Thr463, and corresponding domains from other HIV-1 isolatesappear to range from about 10 to about 11 residues in length.

In selecting the areas of the intervening domains for deletions, it ispreferred to either select those portions showing hypervariability, orareas known to encode epitopes which elicit a significant immuneresponse in vivo. Examples of such epitopes in variable regions of theSF2 isolate include, without limitation, residues 137-158 (V1), residues189-209 (V2), residues 300-327 (V3), residues 367-384 (V3), and residues404-420 (v4). Corresponding epitopes from other isolates are known.Modrow et al., supra. Examples of preferred deletions for SF2, D1-D5,are shown in FIGS. 1 and 2: Thr131-Asn154 (D1), Ser156-His198 (D2),Thr300-His332 (D3), Asn388-Pro414 (D4), and Thr457-Thr463 (D5). Thepolypeptides of the present invention may also have deletions from morethan one variable domain, for example, V1 and V2; V1, V2 and V3; V1, V2and V5; V3, V4 and V5; or V1, V2, V3, V4, and V5.

In a preferred embodiment, the HIV-1 envelope analogs of the presentinvention have deleted therefrom an epitope found in the interveningdomains which is bound by antibodies produced by a mammal infected orimmunized with the particular HIV-1 isolate, or its native envelopeprotein. It is particularly preferred that the deleted epitopes arethose which produce isolate-specific immunodominant responses in amammal. While applicants do not wish to be bound by this theory, it isbelieved that the deletion of these variable epitopes unmasks epitopesin the conserved domains which then become visible to the immune systemby virtue of the absence of the epitopes from the variable domains.

In general, the amino acid sequence according to formula I will rangefrom about 300 to about 850 amino acid residues in length, the actuallength not being critical. The sequence of formula I can be containedwithin a larger polypeptide, for example a fusion protein. Such fusionproteins can include, for example, a fusion between the N-terminalsequence of superoxide dismutase (human or yeast) or beta-galactosidase,where these non-HIV-1 sequences are fused to the N-terminal of the C1domain. Alternatively, non-HIV-1 sequences could also be fused to theC-terminal of the C5 domain. The C1 domain may also be fused to a signalpeptide (e.g., yeast alpha factor, or tpa signal) to provide forsecretion of the HIV-1 envelope analog from a cellular host expressingthe analog.

The HIV-1 envelope muteins of the present invention can be produced byany suitable method, such as direct peptide synthesis or recombinant DNAexpression. The preferred method is to prepare the polypeptides byrecombinant DNA techniques.

The methodology for preparing such recombinant polypeptides is withinthe skill of the art, and the techniques are fully explained in theliterature. See, e.g., Maniatis et al., Molecular Cloning: A LaboratoryManual (1982); DNA Cloning: A Practical Approach, Volumes I & III (D. N.Glover, ed., 1985); Oligonucleotide Synthesis: A Practical Approach (M.J. Gate, ed., 1984); Perbal, A Practical Guide to Molecular Cloning(1984). The production of recombinant HIV-1 polypeptides is known in theart. See, e.g., Luciw et al., (1984) Nature 312:760; Sanchez-Pescador etal., (1985) Science 227:484; Hahn et al., (1984) Nature 312:167; Alizonet al., (1984) Nature 312:757; Ratner et al., (1985) Nature 313:636;Muesing et al., (1985) Nature 313:450; Wain-Hobson et al., (1985) Cell40:9; EPO Pub. Nos. 181,150; 187,041; 227,169; 230,222; and PCT Pub.Nos. WO87/02038; WO87/02989; WO87/04459; WO87/04728. Methods ofrecombinant expression are also disclosed in commonly owned U.S. patentapplication Ser. No. 138,894, filed 24 Dec. 1987, entitled "HumanImmunodeficiency Virus (HIV) Nucleotide Sequences, RecombinantPolypeptides, and Applications Thereof," the disclosure of which isincorporated herein by reference.

To prepare the polypeptides of the present invention by recombinantmethods, a DNA coding sequence for the polypeptides must be provided.Such a coding sequence is a DNA sequence that can be transcribed andtranslated into a polypeptide in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by and include the translation start codon at the 5'(amino) terminus and a translation stop codon at the 3' (carboxy)terminus. DNA sequences encoding the polypeptides of the presentinvention can be prepared either by constructing a synthetic gene fromoverlapping oligonucleotides, or by site-directed mutagenesis of asequence encoding native HIV-1 envelope. See, e.g., Zoller, & Smith(1983) Meth. Enzymol. 100:468-500.

DNA coding sequences for the polypeptides of the present invention canbe maintained on a replicon, which is any genetic element (e.g, plasmid,cosmid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo; i.e., capable of replication under its own control.Vectors are replicons such as a plasmid, phage, or cosmid to which a DNAcoding sequence may be attached so as to bring about the replication ofthe attached segment in vivo.

A host cell is then transformed with a DNA construct containing thecoding sequence under the control of appropriate regulatory sequences inorder to bring about the expression of the DNA coding sequence into thedesired polypeptide. Cellular hosts can include, but are not limited to,bacteria (e.g., E. coli, Bacillus subtilis, B. amyloliquefaciens,Salmonella typhimurium, Klebsiella pneumoniae or Erwinia amylouora),yeast (e.g., Saccharomyces cerevisiae, S. carlsbergensis, S. kluyveri,Kluyveromyces lactis, Pichia, or Schizosaccharomyces), mammalian cells(e.g., CHO cells, COS cells, 293 cells or Xenopus oocytes), and insectcells (e.g., Drosophila embryos or Spodoptera frugiperda). Thetransformed cellular hosts may contain the DNA constructs encoding thepolypeptides of the present invention either on an extrachromosomalelement, or integrated into the chromosome.

Typically, the DNA coding sequence is placed into an expression cassettewhich comprises the DNA coding sequence flanked by appropriateregulatory sequences that control transcription initiation andtermination within the cellular host. Preferably, the expressioncassette contains convenient restriction sites at either end to permitconvenient cloning of the cassette into an appropriate vector fortransformation of the cellular host.

Transcription initiation and termination sequences recognized by thecellular host are DNA regulatory regions which flank a coding sequenceare responsible for the transcription of an mRNA homologous to thecoding sequence which can be translated into the desired polypeptide.Transcription initiation sequences include host promoter sequences,which are DNA regulatory sequences capable of binding RNA polymerase ina cell and initiating transcription of a downstream (3' direction)coding sequence. A coding sequence is "under the control" oftranscription initiation and termination sequences when RNA polymerasebinds the transcription initiation sequences and transcribes the codingsequence into mRNA terminating at the transcription terminationsequence, and the mRNA is then translated into the polypeptide encodedby the coding sequence (i.e., expression).

Cellular hosts transformed with appropriate DNA constructs for theexpression of polypeptides of the present invention are typically grownin a clonal population under appropriate conditions which bring aboutthe expression of the DNA coding sequence of interest. The appropriategrowth conditions will depend upon the cellular host and thetranscriptional and translational regulatory sequences employed. Uponexpression, the recombinant polypeptide is recovered from the culture byany appropriate method, e.g., gel chromatography, immunoabsorption, orgel electrophoresis.

Polypeptides of the present invention can be used as reagents inimmunoassays for detecting the presence in a sample of either anti-HIV-1antibodies or viral antigen. In an immunoassay for viral antigen, forexample, polypeptides according to the present invention can be labeledand used as a competing antigen in a standard competitive ELISA orradioimmunoassay (RIA).

Configurations for immunoassays for antibodies to HIV-1 envelope varywidely, and the present invention contemplates the use of thepolypeptides described herein in any such format. Such formats are wellknown in the art. See, e.g., Immunoassay: A Practical Guide (P. W. Chan& M. T. Perlstein, eds., 1987); McDougal et al., (1985) J. Immunol.Meth. 76:171-183; U.S. Pat. Nos. 4,629,783; 4,281,061; 4,520,113;4,591,552; 4,134,792 (incorporated by reference herein). Whether theassay format is homogeneous or heterogeneous, and the measurement methodis direct or indirect (e.g., competition), all such immunoassays havethree common steps. First, a liquid sample suspected of containing theantibodies is provided. This sample is then contacted with polypeptidesaccording to the present invention under conditions which will allow anyantibodies having an epitope on the polypeptides to become boundthereto. Finally, there is a detecting step wherein it is determinedwhether or not any antibody bound to the polypeptide.

A preferred format for an anti-HIV-1 antibody assay is a heterogeneousimmunoassay in which the polypeptides of the present invention areimmobilized on, for example, a solid support. The selection of theappropriate solid support for immobilization of the polypeptide isconventional and within the skill of the art. In a typical assay, thesample suspected of containing the antibodies is contacted with theimmobilized polypeptide and allowed to incubate under the appropriateconditions. The immobilized polypeptide is then separated from thesample and washed to remove any unbound antibody. The detecting step canconstitute, for example, contacting the washed, immobilized polypeptidewith an antibody that will recognize an epitope located on theanti-HIV-1 antibodies (i.e., anti-xenogenic), this second antibody beingappropriately labeled for detection (e.g., radiolabeled, enzymeconjugated, avidin/biotin). After an appropriate incubation and washingstep, the immobilized polypeptide is then assayed for the presence ofthe labeled second antibody. Alternatively, a competition immunoassaycan be used where a labeled reference antibody to the immobilizedpolypeptide is incubated along with the sample suspected of containingthe anti-HIV-1 antibodies, and the presence of such antibodies aredetermined by measuring a reduction or inhibition of binding of thelabeled reference antibody to the immobilized polypeptide. See, e.g.,PCT Pub. No. WO87/07957.

Kits suitable for immunodiagnosis and containing the appropriate labeledreagents are constructed by packaging the appropriate materials,including the polypeptides of the present invention and any antibodiesused in the assay, in suitable containers along with remaining reagentsand materials required by the assay format; e.g., incubation media, washmedia, and means for measuring the presence of the analyte (e.g.,enzyme-labeled antibodies and enzyme substrate). Other labels useful inthe practice of immunoassays according to the present invention includeradioisotopes and fluorescing compounds.

Polypeptides according to the present invention can be employed toselectively raise antibodies in a mammal to epitopes found in theconstant domains of gp120env or gp160env. For example, parenterallyadministering polypeptides of the present invention to a mammal willcause an immune reaction in the animal, thereby producing antibodies toepitopes found in the conserved domains. Such antibodies can berecovered to make polyclonal antiserum, or antibody-producing cellsrecovered for fusion (or another immortalization technique) to producemonoclonal antibody-producing cell lines. See, e.g., M. Schreier et al.,Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodiesand T Cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies(1980); U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887.Antiserum produced by the above method will be advantageous in that itwill possess a high titer of antibodies which are reactive to all ormost HIV-1 isolates. In a similar manner, the screening of hybridomasfor non-isolate-specific antibodies will be facilitated by theelimination of some or all of the immunodominant epitopes of thehypervariable regions.

To generate such an antibody response, polypeptides of the presentinvention are typically formulated with a pharmaceutically acceptablecarrier for parenteral administration. The formulation of suchcompositions, including the concentration of the polypeptide and theselection of the vehicle and other components, is within the skill ofthe art.

A pharmaceutically acceptable vehicle, suitable for parenteralinjection, is usually nontoxic and nontherapeutic. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, andHank's solution. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Parenteral vehicles mayalso take the form of suspensions containing viscosity-enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. The vehiclewill also usually contain minor amounts of additives, such as substancesthat enhance isotonicity and chemical stability. Examples of buffersinclude phosphate buffer, bicarbonate buffer and tris buffer, whileexamples of preservatives include thimerosal, m- or c-cresol, formalinand benzyl alcohol. The muteins of the present invention may also beformulated into liposomes for parenteral administration. Standardformulations will either be liquid injectables or solids which can betaken up in a suitable liquid as a suspension or solution for injection.Thus, in a nonliquid formulation, the vehicle may comprise dextrose,human serum albumin, preservatives, etc., to which sterile water orsaline could be added prior to administration.

Various adjuvants are known in the art which can also be employed in thevaccine formulations of the present invention; e.g., Freund's adjuvant,avridine, aluminum salts Al(OH)₃ AlPO₄, Al₂ (SO₄)₈ !, Ca₃ (PO₄)₂,saponin, DDA, Plusonics, oil-in-water emulsions (containing, e.g.,avridine, dextran sulfate or vitamin E), water-in-oil emulsions(containing, e.g., polysorbate 8), and muramyl peptides (e.g., di- andtripeptides in carriers such as oil-water emulsions or liposomes). Theselection of the appropriate adjuvant and its concentration in thevaccine composition is within the skill of the art.

Many protocols for administering the vaccine composition of the presentinvention to animals are within the skill of the art. The preferredroute of administration is parenteral, particularly intramuscular,although administration may also be intravenous. The concentration ofthe polypeptide antigen in the vaccine composition is selected so thatan effective dose is presented to the host mammal (e.g., primate) toelicit antibodies to the polypeptide's epitopes. Within wide limits, thedosage is not believed to be critical. Typically, the vaccinecomposition is administered in a manner which will deliver between about1 to about 1,000 ug of the polypeptide antigen in a convenient volume ofvehicle (e.g., about 1-10 ml). Preferably, the dosage in a singleimmunization will deliver from about 1 to about 500 ug of polypeptideantigen, more preferably about 5-10 to about 100-200 ug (e.g., 10-100ug). It may also be preferred, although optional, to administer asecond, booster immunization to the mammal several weeks to severalmonths after the initial immunization. It may be helpful to readministera booster immunization to the mammal once every several years tomaintain high antibody titer.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the claims in any way.

EXAMPLES I

The following example describes the construction of DNA sequencesencoding HIV-1 SF2 envelope muteins according to the present invention.The hypervariable regions and the corresponding deletions are shownbelow in Table 1.

                  TABLE 1                                                         ______________________________________                                        Hypervariable Regions and Deletion Mutants                                                           Corresponding                                          Hypervariable Regions  Deletion Mutant(s)                                     ______________________________________                                        V1    131 through 154  D1    131 through 154                                  V2    156 through 198  D2    156 through 198                                  V3    292 through 365  D3    300 through 332                                  V4    388 through 414  D4    388 through 414                                  V5    456 through 465  D5    457 through 463                                  ______________________________________                                    

DNA encoding sequences for muteins of the present invention wereprepared by mutagenesis of a gp120env SF2 gene. Mutagenesis was achievedemploying the procedure described by Zoller and Smith, (1983) Meth.Enzymol. 100:468-500, modified as described below. Synthetic DNAmutagenesis and sequencing primers (Table 2) were prepared by automatedoligonucleotide synthesis on a silica support as described by Urdea etal., (1983) Proc. Natl. Acad. Sci. USA 80:7461-7465, usingN,N-diisopropyl phosphoramidites. Sequencing primers were designed forsequencing by the dideoxynucleotide chain termination method inbacteriophage M13. Sanger et al., (1977) Proc. Natl. Acad. Sci. USA74:5463. The sequencing primers were designed to be complementary toM13mp8 recombinant templates, of 18 bases in length, and to anneal at aposition at least 50 bases away from the mutation locus. Table 2indicates the oligomers employed for mutagenesis screening andsequencing.

The mutagenesis template contained the entire plasmid pSV7dARV120tPA,which contains the SF2 gp120env gene coupled to the tPA signal sequence(described below). The plasmid was linearized at its unique PstI siteand cloned into the unique PstI site of M13mp8.

                                      TABLE 2                                     __________________________________________________________________________    Mutagenesis Primers                                                           D1      5' CCACTCTGTGTTACTTTAAATTGCTGCTCTTTCAATATCACCACAAGC 3'                D2      5' ATAAAAGGAGAAATAAAAAACTGCTGTAACAGATCAGTCATTACACAG 3'                D3      5' AATGAATCTGTAGCAATTAACTGTTGTAACATTAGTAGAGCACAATGG 3'                D4      5' GGGGAATTTTTCTACTGTTGTAGAATAAAACAAATTATAAACATGTGG 3'                D5      5' CTGCTATTAACAAGAGATGGTGGTGAGGTCTTCAGACCTCGAGGAGGA 3'                D1 + D2 5' CCACTCTGTGTTACTTTAAATTGCTGCTGTAACAGATCAGTCATTACA 3'                DNA Sequencing Primers                                                        D1, D2, D1 + D2                                                                       5' TAATCAGTTTATGGGATC 3'                                              D3      5' CTGTTAAATGGCAGTCTA 3'                                              D4, D5  5' CAATCCTCAGGAGGGGAC 3'                                              Screening Probes                                                              D1      5' TTAAATTGCTGCTCTTTC 3'                                              D2      5' AAAAACTGCTGTAACAGA 3'                                              D3      5' AATTAACTGTTGTAACATTA 3'                                            D4      5' GGGGAATTTTTCTACTGTTGTAGAATAAAACAAATTATAAACATGTGG 3'                D5      5' ATGGTGGTGAGGTCTT 3'                                                D1 + D2 5' CCACTCTGTGTTACTTTAAATTGCTGCTGTAACAGATCAGTCATTACA                   __________________________________________________________________________            3'                                                                

Mutagenesis of M13/pSV7dARV120tPA recombinants was performed usingpurified templates by annealing and extending the appropriate primerwith the Klenow fragment of DNA polymerase I. Individual deletionmutants D1, D2, D3 and D5 were obtained utilizing the appropriatemutagenesis primer in a single round of mutagenesis (see Table 2).Deletion mutant D4 was obtained utilizing the appropriate mutagenesisprimer with the annealing and elongation reactions performed at 37° C.and in the presence of 125 ug/ml gene 32 protein. Combination deletionmutant D1+D2 was obtained utilizing the appropriate mutagenesis primerusing a template derived from deletion mutant D1.

Following transfection of JM101 cells (Zoller and Smith, supra), plaqueswere grown at a density of 200-1,000/plate and lifted onto filters andscreened by hybridization with the appropriate mutagenesis primer orprobe (see Table 2).

The DNA sequence of putative positive clones was determined usingsuitable primers and template preparations. Once the mutagenized locusand flanking segments (i.e., at least 50 bases) were confirmed by DNAsequence analysis, replicative form (RF) DNAs were digested by PstIrestriction endonuclease and the entire mutagenized mammalian expressionvector pSV7dARV120tPA containing the deletion was recovered. The vectorprovides an SV40 early promoter and enhancer for expression of SF2gp120env gene, SV40 polyadenylation site, and an SV40 origin ofreplication for use of the vector in COS cells.

Following recovery of pSV7dARV120tpa plasmids for wild-type gp120 or fora deletion mutant, the plasmid was digested with SalI to excise thecomplete gene spliced to the human tPA 5' untranslated sequences andsignal sequences. The SalI fragment was subcloned into the unique SalIcloning site of the mammalian cell expression vector pCMV6a. Theresulting plasmid was screened to verify the correct orientation of thegene with respect to the promoter and polyadenylation signals, and thisplasmid was named pCMV6ARV120tpa for the wild-type gp120 sequences. Incases where the pSV7d vector was not recovered directly as a plasmid,the SalI fragment containing the gene was subcloned from the M13mutagenesis template clone into pCMV6a.

Combination deletion mutant D4-D5 was obtained utilizing the appropriateD5 mutagenesis primer using a template derived from deletion mutant D4.

Combination deletion mutant pSV7d120D3-D4-D5 was obtained by subcloningthe region containing D4-D5 by digestion of M13pSV7d120D4-D5 with MstIIand HindIII and insertion of this 334 bp fragment into MstII and HindIIIdigested pSV7d120D3. The SalI fragment from pSV7d120D3-D4-D5 containingD3-D4-D5 was subcloned into the SalI site of pCMV6a to createpCMV6a120D3-D4-D5.

Combination deletion mutant D1-D2-D5 was obtained by subcloning theregion containing D1-D2 by digestion of pCMV6a120D1-D2 with NheI andBglII and insertion of this 540 bp fragment into NheI and BglII digestedpCMV6a120D5.

Combination deletion mutant D1-D2-D3-D4-D5 was obtained by subcloningthe fragment containing the D1-D2 region by digestion ofM13pSV7d120D1-D2 with NheI and BglII and insertion of this 539 bpfragment into NheI and BglII digested pCMV6a120D3-D4-D5.

Expression vector pCMV6a can be regenerated by excising the codingsequence for gp120 from pCMV6ARV120tpa with SalI. The mutein codingsequences described above can all be constructed from the wild-typegp120 coding sequence in pCMV6ARV120tpa as described for pSV7dARV120tpa.Table 3 sets forth the names and deletions of the various M13-pSV7d- andpCMV6a-based vectors made according to the above protocol.

                  TABLE 3                                                         ______________________________________                                        Gene               pSV7d        pCMV6a                                        Version                                                                             M13 Vector   Vector       Vector                                        ______________________________________                                        Wild- M13pSV7d120  pSV7dARV120tpa                                                                             pCMV6ARV120tpa                                type                                                                          D(1 + M13pSV7d120D1 +                                                                            not made     pCMV6a120D1 + 2                               2)    2                                                                       D1    M13pSV7d120D3                                                                              pSV7d120D1   pCMV6a120D1                                   D2    M13pSV7d120D2                                                                              pSV7d120D2   pCMV6a120D2                                   D3    M13pSV7d120D3                                                                              PSV7d120D3   pCMV6a120D3                                   D4    M13pSV7d120D4                                                                              not made     pCMV6a120D4                                   D6    M13pSV7d120D5                                                                              not made     pCMV6a120D5                                   D3 +  M13pSV7d120D3 +                                                                            not made     not made                                      D4    D4                                                                      D4 +  M13pSV7d120D4 +                                                                            pSV7d120D4 + D5                                                                            not made                                      D5    D                                                                       D3 +  not made     pSV7d120D3 + pCMV6a120D3 +                                 D4 +               D4 + D5      D4 + D5                                       D5                                                                            D(1 - not made     not made     pCMV6a120D1 - 5                               5)                                                                            D(1 + not made     not made     pCMV6a120D(1 +                                2 + 5)                          2 + 5)                                        ______________________________________                                    

pSV7dARV120tPA (FIG. 3) was constructed as follows. An env gene wasmodified by in vitro mutagenesis to eliminate any potentialtransmembrane domains and to provide a stop codon following theprocessing site between the gp120 and gp41 domains of the gp160 protein.This mutagenesis was accomplished by subcloning the fragment whichencodes the env gene from clone pSV7c/env (ATCC Accession No. 67593) byexcising with HindIII and XhoI (SF2 clone positions 5582 and 8460) andinserting the 2.8 kb fragment into M13mp19 previously digested withHindIII and SalI. A 37 bp oligonucleotide of the following sequence wasused to alter the sequence at the gp120/gp41 processing site at position7306 to encode 2 stop codons and two restriction endonuclease sites.

5'-GAACATAGCTGTCGACAAGCTTCATCATCTTTTTTCT-3'

The sequence of the wild-type gene and the mutant are shown below:

Wild-type sequence: ##STR1##

Following mutagenesis, the gene was engineered for optimal secretioninto the medium. A 268 bp XbaI-NdeI fragment containing the heterologous5' untranslated sequences and signal sequences from human tPA fused tothe 5' end of env was excised from the M13 clone M13tpaS.NheIenvdescribed below. This fragment was ligated with a 1363 bp NdeI-SalIfragment encoding the remainder of the gp120 coding region which wasisolated from the gp120 mutant (positions 5954 and 7317) describedabove, and both fragments were inserted into the vector pSV7d (describedbelow) previously digested with XbaI and SalI.

Expression vector pSV7d can be generated by digesting pSV7c/env withBglII and XbaI, and then ligating the digested plasmid with thefollowing linker: ##STR2##

M13tpaS.NheIenv was constructed as follows. The 5' end of the env codingsequence was modified to accept a heterologous signal sequence known todirect efficient secretion of both the homologous gene (human tissueplasminogen activator) and deletion variants of this gene. van Zonnefeldet al., (1986) Proc. Natl. Acad. Sci. USA 83:4670.

A portion of the HIV-1 SF2 gene in a lambda phage (ATCC Accession No.40143) was excised with SacI and StuI (positions 5555 and 6395) and wassubcloned into the vector M13mp19 Yanisch-Perron et al., (1985) Gene33:103-109! between SacI and SmaI. Oligonucleotide-directed mutagenesisZoller et al., (1983) Meth. Enzymol. 100:468-500! was used to create anNheI site at the junction of the natural signal peptide and the matureenvelope polypeptide using the following oligonucleotide:

5'-GATGCTCTGTTCAGCTAGCGAAAAATTGTGG-3'

This mutagenesis changes cytosine-5867 to guanine and adenine-5868 tocytosine, thereby creating an NheI site and altering the codon forthreonine-30 to code for serine.

In parallel, the tPA gene was likewise mutagenized in M13 to place anNheI site near the carboxyl end of the tPA signal peptide. The followingsequences show the 5' UT sequences and signal for wild-type tPA leaderand for the NheI variant.

Wild-type sequence of the tPA signal: ##STR3##

The NheI variant of the tPA signal: ##STR4##

Following mutagenesis and sequence verification, a 174 bp fragmentcontaining 99 bp of 5' untranslated sequence and the signal sequencefrom tPA was excised from the tPA-containing M13 clone using SalI andNheI and fused to the 559 bp fragment containing the 5' end of the envgene which was excised from the env-containing M13 clone with NheI andHindIII (contributed by the M13 polylinker), and these fragments weresubcloned into M13mp18 between SalI and HindIII to give plasmidM13tpaS.NheIenv. The DNA and amino acid sequence of the tPA signal fusedto the 5' end of env gene is: ##STR5##

Example II

This example describes the expression of HIV-1 env analogs in mammaliancells.

DNA encoding the complete env gene with the substituted tPA signalsequence was excised from the plasmid pSV7dARV120tpa using therestriction endonuclease SalI and inserted into the unique SalI site ofthe mammalian cell expression vector pCMV6a, and the resulting plasmidDNA was screened to verify the correct orientation of the gene withrespect to the promoter and polyadenylation signals (see Example I). Theresulting plasmid, pCMV6ARV120tpa, and the plasmid pSV7dARV120tpa wereused to transfect COS-7 cells to test expression and secretion of gp120into the medium. pCMV6ARV120tpa was at least 50-fold more efficient inexpressing gp120 compared with the pSV7dARV120tpa expression plasmid inthese cells.

Permanent cell lines were isolated as follows: human kidney 293 cellswere plated at a density of 50-70% confluency in DME supplemented withglutamine (292 mg/L), sodium pyruvate (110 mg/L), glucose (4.5 g/L),penicillin (1000 U/L), streptomycin (1000 U/L), 3.7 g/L sodiumbicarbonate, and fetal calf serum (10% v/v). Cells were exposed to acalcium phosphate coprecipitate following standard techniques with 10 ugeach of the HIV env expression plasmid pCMV6ARV120tpa (wild type ordeletion mutant) and a plasmid encoding the selectable markerneomycin-resistance, pSV2neo (Ref), for six hours at 37° C. in a 10% CO₂atmosphere. Cells were washed and exposed to a 3 to 4 minute shock of15% DMSO or glycerol in HEPES-buffered saline, and growth medium(described above) was replaced for 48 hours. Trypsinized cells werereplated at a lower density in 400 ug/ml G418 (Sigma) in DMEsupplemented as above. Colonies grew to the 100 cell per focus stage inone week to ten days, and these colonies were transferred individuallyto 96 well plates. Clones were screened for gp120 production by testingthe conditioned cell medium using an ELISA described below. Positiveclones were scaled up to T75 flasks, aliquots of cells frozen, and cellsupernatants were collected for further characterization, e.g. CD4binding.

Alternatively, CHO dhfr- cells plated at a density of 50-70% confluencewere cotransfected by calcium phosphate coprecipitation using 10 ug eachof the plasmids pCMV6ARV120tpa (or analogous deletion mutant expressionvector) and the selectable marker dhfr encoded in the plasmid pAd-dhfr.Following exposure to the coprecipitate and shock solutions as describedabove, cells were incubated for 48 hours in Ham's F12 supplemented withglutamine (292 mg/L), sodium pyruvate (110 mg/L), sodium bicarbonate(3.7 g/L), glucose (4.5 g/L), penicillin (1000 U/L), streptomycin (1000U/L), proline (150 mg/L), and fetal calf serum (10%). Forty-eight hoursafter transfection, cells were plated at a density of approximately onetenth in DME supplemented as described for F12 above, except that thefetal calf serum was replaced with dialyzed fetal calf serum (10%).Colonies were transferred individually to 96 wells after about twoweeks, then screened using the ELISA assay for gp120 secreted into themedium. Positive clones were scaled up as described above.

For detection of gp120 or gp120 hypervariable region deletion mutants inthe supernatants of COS, CHO, or 293 cells, conditioned medium wasassayed by ELISA specific for gp120 sequences. Pooled HIV-positive humanserum inactivated by treatment with psoralen was affinity purified onStaphylococcus Protein A Sepharose by standard techniques. This serumwas coated on Immunlon 1 96 well ELISA plates at a concentration of 5ug/ml in PBS and plates were incubated 12 hours to two months at 4° C.Following incubation, plates were washed as described for the titrationELISA (Example IV), and samples and standards (including purifiedrecombinant HIV-1 gp120env from yeast) were applied to the plate intwo-fold dilution series using the dilution buffer described for thetitration ELISA. The range of the assay is 100 ng/ml to 200 pg/ml.Samples were incubated for 12 hours at 4° C. Samples were aspirated andplates were washed as above. Samples were then incubated with anappropriate dilution of rabbit serum from rabbits immunized withrecombinant SF2 gp120env analog (usually 1:100 dilution of Protein ASepharose affinity-purified serum in dilution solution) for 1 hour at37° C., followed by washing. Color development was with ABTS, asdescribed for the titration ELISA. Plates were read as described, andthe amount of gp120 in each sample was determined by using a standardcurve derived from the standard on the same plate. The assay wasverified by showing that HIV-infected HUT 78 cells (infected celllysate) gave a positive signal, while uninfected cell lysates werenegative.

Example III

This example describes the recombinant production of muteins in yeasthosts according to the present invention.

The starting plasmid used for the construction of yeast expressionvectors was plasmid pJS150. This plasmid is similar to plasmidpBS24.1/SOD-SF2env4-5 (U.S. Ser. No. 138,894, filed 24 Dec. 1987,supra), and has had the yeast promoter and HIV coding sequences locatedbetween the unique BamHI and SalI sites replaced with a yeast ADH2/GAPDHpromoter and a portion of an Zairan HIV-1 isolate envelope codingsequence. In addition, the NheI restriction site located in the plasmidvector portion was destroyed by cutting with NheI, nuclease S1treatment, followed by ligation.

Plasmid pJS150 was digested with restriction enzyme NcoI, which cutsjust after the translation initiation ATG codon downstream from theADH2/GAPDH promoter (as well as at other sites in the vector). Thefragments were ligated to an NcoI/NheI adaptor having the sequence:

5'-CATGGCTAGCCCCCGATCGGGG-5'

After ligation, the DNA sequences were digested with BamHI and NheI togenerate a 1.2 kb BamHI-NheI fragment containing the ADH2/GAPDH promoterand an NheI sticky end immediately downstream. This DNA fragment wasisolated by gel electrophoresis and is referred to as Fragment A.

A second digest was performed by cutting plasmid pJS150 with BamHI andSalI to generate a 13 kb linear DNA vector. This DNA fragment was alsopurified by gel electrophoresis, and is referred to as Fragment B. Athird DNA fragment containing a coding sequence for gp120 D3 mutein wasisolated from pCMV6a120D3 (Table 3 of Example I) by digestion with NheIand SalI. The approximate 1.2 kb coding fragment was then gel isolated,and is referred to as Fragment C.

The three fragments (A, B and C) were then ligated together, and theresulting ligation mix was used to transform E. coli strain HB101 toampicillin resistance. A plasmid containing all three fragments in theproper orientation is shown in FIG. 5. This D3 yeast expression vectorwas designated pHL15. Expression vectors for additional muteins werealso constructed by cloning the mutein-encoding sequence of thepCMV6a-based vectors (Example I, Table 3) into NheI/SalI-digested pHL15,thereby replacing the D3 coding sequence. The deletions andcorresponding vectors that were made are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                               Deletion       Vector                                                  ______________________________________                                               D1             pHL24                                                          D2             PHL25                                                          D3             pHL15                                                          D4             pHL26                                                          D5             pHL27                                                          D1 + D2        pHL22                                                          D3 + D4 + D5   pHL21                                                          D1 through D5  pHL20                                                   ______________________________________                                    

The yeast expression vectors described above were used to transformSaccharomyces cerivisiae strain JSC308 (ATCC accession No. 20879,deposited 5 May 1988) to uarcil prototrophy. Uracil prototrophs werethen streaked onto leucine selective plates to isolate leucineprototrophs (as a result of plamsmid amplification in vivo). Expressionof the deletion muteins was achieved by growing a seed culture of theleucine prototrophs in leucine selective medium and then diluting itinto approximately 10 liters of a rich medium containing yeast extract,peptone, and glucose. Either 2% or 4% glucose was used as the carbonsource in the media, whichever appeared optimal.

Muteins were purified as follows. Frozen yeast cells were thawed andsuspended in 1 volume of lysis buffer, 0.001M PMSF, 0.001M EDTA, 0.15MNaCl, 0.05M Tris-HCl pH 8.0), and 1 volume of acid-washed glass beadsadded. Cells were broken in a noncontinuous system using a 300 ml glassunit of Dyno-mill at 3000 rpm for 10 min. the jacket was kept cool by a-20° C. ethylene glycol solution. Glass beads are decanted by lettingthe mixture set for 3 min on ice. The cell extract was recovered andcentrifuged at 18,000 rpm (39,200×g) for 35 min. The supernatant wasdiscarded and the precipitate (pellet 1) further treated as indicatedbelow.

Pellet 1 was resuspended in 4 volumes of Tris-HCl buffer (0.01MTris-HCl, pH 8.0, 0.01M NaCl, 0.001M PMSF, 1 ug/ml pepstatin, 0.001MEDTA, 0.1% SDS) and extracted for 1 hr at 4° C. with agitation. Thesolution was centrifuged at 6,300×g for 15 min. The insoluble fraction(pellet 2) was resuspended in 4 volumes (360 ml) of PBS (per liter: 0.2g KCl, 0.2 g KH₂ PO₄, 8.0 g NaCl, 2.9 g Na₂ HPO₄.12H₂ O), 0.1% SDS,0.001M EDTA, 0.001M PMSF, 1 ug/ml pepstatin, and centrifuged at 6,300×gfor 15 min. This pellet (pellet 3), was suspended in 4 volumes of PBS,0.2% SDS, 0.001M EDTA, 0.001M PMSF, 1 ug/ml pepstatin and extracted for12 hr at 4° C. with agitation on a tube rocker. The solution was thencentrifuged at 6,300×g for 15 min. The soluble fraction was recoveredfor further purification as indicated below. (The pellet can bereextracted by resuspending it in 4 volumes of 2.3% SDS, 5%beta-mercaptoethanol, and boiling for 5 min. After boiling, the solutionis centrifuged at 6,300×g for 15 min. The soluble fraction is recoveredfor further purification.)

The soluble fraction was concentrated by precipitation with 30% ammoniumsulfate at 4° C. The pellet (pellet 4) was then resuspended in 2.3% SDS,5% beta-mercaptoethanol, and chromatographed on an ACA 34 or ACA 54 (LKBProducts) gel filtration column (depending on the size of the mutein).The column was equilibrated with PBS, 0.1% SDS, at room temperature.Chromatography was developed in the same solution with a flow rate of0.3 ml/min. If needed, pooled fractions were concentrated by vacuumdialysis on Spectrapor #2 (MW cutoff 12-14K).

Example IV

This example describes an immunoassay for anti-HIV antibodies employingHIV-1 env analogs.

Immulon-1 96 well immunoassay plates are coated with gp120 antigen bydispensing 100 ul per well of a 2 ug per ml purified antigen produced inyeast (Env SF2 wild type, env HTLV wild type, env Zr6 wild type, envSF2-D3, env SF2-D1-D2, env SF2-D3-D4-D5, env SF2-D1-D2-D3-D4-D5 in 50 mMborate pH 9.2 at 4° C. for at least 12 hours and less than 60 days. Thecoating is aspirated from the plate, and the plate is washed six timesby dunking in a solution of 0.137M NaCl (0.8%), 0.05% Triton-X 100. Theplate is patted dry, and 100 ul per well of 100 mM sodium phosphate,0.1% casein, 1 mM EDTA, 1% Triton-X 100, 0.5M NaCl, 0.01% thimerosol pH7.5 (dilution solution) is added.

Sera to be tested are prepared for analysis by diluting 5 ul test serum(from HIV-positive humans and normal humans, or from immunized animals)in 500 ul of the dilution solution above (1/100 dilution v/v). Thesolution in the top wells is aspirated off and 150 ul of diluted testserum is added. Using a multichannel pipettor set at 50 ul, dilutionsdown each column are carried out, taking 50 ul each time (1/3 dilutionv/v). The plates are then incubated 1 hour at 37° C. with the platewrapped in plastic wrap. The samples are then aspirated off and washed 6times as above. Then, 100 ul per well of goat anti-human IgG conjugatedto horseradish peroxidase (Tago 2733 Lot 330102) diluted 1/2000 indilution solution is added. The plates are then incubated 30 minutes at37° C. covered in plastic wrap. The solution is again aspirated off andwashed 6 times as above. 100 ul per well of color developing solution isadded 100 ul ABTS stock (15 mg/ml 2,2'-A zino-di-(3 ethylbenzthiazolenesulfonic acid), Sigma A-1888, in water, stored in the dark at 4° C.)plus 3.3 ul 30% hydrogen peroxide in 10 ml citrate buffer (10.5 g citricacid per liter water, pH to 4.0 with 6M NaOH)!. The ABTS solution ismade no more than 10 minutes prior to use, and the solution should bemade with citrate buffer at room temperature. Plates in plastic wrap areincubated at 37° for 30 minutes in the dark. The reaction was stoppedwith 50 ul per well of 10% sodium dodecyl sulfate.

Plates were read at 415 nm with a reference wavelength at 600 nm. Titersare determined as follows from the raw data: absorbance (linear axis) isplotted vs. dilution factor (log axis) on semilog paper or computerprogram.

Included in the test sera are standard reference sera as positivecontrols. For titration of human sera, HIV-positive serum 20058 from theInterstate panel was used; for goat sera, a goat serum developed byimmunization with envSF2 wild-type, reference 02GT097.2 was used; andfor titration of guinea pig sera, a standard reference guinea pig serumwas used, likewise obtained by immunization with envSF2 wild-type,reference +Gp sera 1935/77. From the values obtained on the ELISAreader, an absorbance value was chosen that is half-maximal (OD₅₀)(between 0.5 and 0.7) in the linear portion of the standard curve(positive control), for each plate. The average titer for the standardson the plate were determined. The average titer was divided by the"Reference Titer" for that species and antigen plate, and the resultingnumber is the "correction factor". The test sera titers were divided bythe correction factor to yield the adjusted, normalized titers.

A panel of selected human sera was tested to determine their titers onall of the recombinant antigens, including the deletion muteins env SF2D1-2, env SF2-D-3, env SF2-D1-2-3-4-5, and env SF2-D3-4-5. Results areshown in FIG. 6 (North American serum panel) and FIG. 7 (African serumpanel). The graphs show that for these serum samples, the recombinantantigen env SF2-D1-2 is as efficient or more efficient in detecting seraas is env SF2 wild-type. Recombinant antigens env SF2-D3, env SF2-D3-4-5and env SF2-D1-2-3-4-5 are as efficient as env HTLVIII wild-type or envZr6 wild-type.

Example V

This example describes the immunization of mammals with recombinanthypervariable region muteins and the detection of immune responses inthese animals in response to these injections. Recombinant env antigenspurified from yeast were used to generate anti-HIV antibodies inexperimental animals of very high titer. In both goats and guinea pigs,the titers of the resulting sera were at a similar level, whether theimmunogen was env SF2 wild-type or an env SF2 hypervariable mutein.

Immunization of Guinea Pigs

In order to test if guinea pigs imunized with env SF2 muteins derivedfrom yeast were capable of generating a strong immune response, theseanimals were immunized with several muteins and control antigens.

For each antigen, six Hartley guinea pigs were immunized in the footpadwith 50 ug each antigen mixed with 50 ug adjuvant (see below) at threeweek intervals with a total of seven injections. Blood samples weretaken at the time of each injection (prebleed at injection 1), and theserum was monitored by titration in the ELISA described above in ExampleIV for the production of antibodies directed against the immunizingantigen and against heterologous env antigens.

Antigens:

Env SF2-D3

Env SF2-D-1-2-3-4-5

Env SF2-D3-4-5

Env HTLVIII wild-type

Adjuvant:

Muramyl-tripeptide-phosphatidyl ethanolamine in squalene-Tween.

Vaccine:

Mix adjuvant with correct volume of 10X Squalene-Tween (carrier) to makea final concentration of 1X (4% squalene, 0.008% Tween-20), antigen, andPBS to make the correct volume and dose (50 ug each of antigen andadjuvant per animal in 100 ul injection volume per animal. Warm themixture for 5 min at 45° C. Pass the warm mixture in and out of a 23 ga.needle six times, taking care to avoid introducing air into the mixture.Inject into the animals immediately. If the injection procedure takesmore than 5 min, remix the emulsion by shaking by hand every fewminutes.

Control:

Control animals receive the adjuvant in carrier.

Results:

The results of the ELISA are shown in FIG. 8. As can be seen, themuteins of the present invention are as effective as wild-type envelopein generating high antibody titers. A standard virus neutralizationassay was also conducted the guinea pig sera. The results are shown inTable 5. The data show that deletion mutants of the present inventioncan generate significant levels of neutralizing antibodies.

                  TABLE 5                                                         ______________________________________                                        Virus Neutralization by Guinea Pig Sera                                                   Animal    ELISA Titer/                                                                            Neutralization                                Antigen     Number    env SF2   Titer HIV-SF2                                 ______________________________________                                        Env SF2 D3  2477      280,000   50                                            (COOH half) 2476      221,000   500                                                       2479      274,000   250                                                       2480      190,000   100                                                       2481      115,000   <20                                           Env SF2 D3  2471      45,000    >500                                                      2473      57,000    <20                                                       2474      116,000   <20                                                       2475      26,000    <20                                                       2476      16,000    <500                                          Env SF2 D(3-5)                                                                            2489      342,000   500                                           (full length)                                                                             2490      88,000    30                                                        2491      131,000   45                                                        2492      83,000    25                                                        2494      253,000   30                                            Env SF2 D(1-5)                                                                            2483      131,000   <20                                                       2484      71,000    <20                                                       2485      60,000    <20                                                       2486      54,000    <20                                                       2487      100,000   <20                                                       2488      133,000   <20                                           Env HTLVIII 2523      23,000    <20                                                       2524      37,000    <20                                                       2525      52,000    <20                                                       2526      23,000    <20                                                       2527      70,000    <20                                                       2528      14,000    <20                                           ______________________________________                                    

Immunization of Goats

In order to test if goats immunized with env SF2 muteins derived fromyeast were capable of generating a strong immune response, these animalswere immunized with several muteins and control antigens.

For each antigen, two goats were immunized intramuscularly with 1 mgeach primary injection (complete Freund's adjuvant) and 0.5 mg eachbooster injection (incomplete Freund's adjuvant) at three week intervalswith a total of six injections. Blood samples were taken at the time ofeach injection (prebleed at injection 1), and the sera were monitored bytitration in the ELISA assay described above in Example IV for theproduction of antibodies directed against the immunizing antigen andagainst heterologous env antigens.

    ______________________________________                                        Antigens:   Env SF2-D3       Lot 3064/1a                                                  Env SF2-D1-a2-3-4-5                                                                            Lot 3064/5a                                                  Env HTLVIII wild-type                                                                          Lot 3064/15a                                     ______________________________________                                    

Adjuvant: Complete Freund's and incomplete Freund's.

Vaccine: Mix 0.5 ml antigen (1 mg) with 0.5 ml complete Freund's.Emulsify by standard procedures and inject. For boosters, mix 0.5 mlantigen (0.5 mg) with 0.5 ml incomplete Freund's. Emulsify and inject.

Control: Control animals receive the adjuvant in carrier.

Results: The results of the ELISA are shown in FIG. 9. As can be seen,the muteins of the present invention are at least as effective aswild-type polypeptides in antibody titer levels generated.

Deposit of Biological Materials

Vectors pCMV6ARV120tpa and pHL15, both in E. coli HB101, were depositedwith the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md., USA, on 13 Sep. 1988, and will be maintained under theprovisions of the Budapest Treaty on the Deposit of Microorganisms. Theaccession number for the pCMV6ARV120tpa deposit is 67792, and theaccession number for the pHL15 deposit is 67793.

These deposits are provided for the convenience of those skilled in theart, and are neither an admission that such deposits are required topractice the present invention, nor that equivalent embodiments arebeyond the skill of the art in view of the present disclosure. Theavailability of these deposits is not a grant of any license (e.g., tomake, use or sell the deposited materials) under this or any otherpatent. The nucleic acid sequences of the deposited materials areincorporated in the present disclosure by reference and are controllingif in conflict with any sequence described herein.

Although the foregoing invention has been described in some detail byway of illustration and example, it will be obvious that changes andmodifications may be practiced within the scope of the apended claims.

I claim:
 1. An immunoassay method of detecting antibodies to humandeficiency virus type 1 (HIV-1), comprising:(a) providing a liquidsample to be tested for the presence of anti-HIV-1 antibodies; (b)contacting said sample with a human immunodeficiency virus type 1(HIV-1) envelope mutein having the structure C₁ -V₁ -V₂ -C₂ -V₃ -C₃ -V₄-C₄ -V₅ -C₅ wherein said mutein retains the conserved domains C₁ -C₅ andhas a deletion of at least one of the hypervariable domains V₁ -V₅ ; and(c) detecting antibody bound specifically to said polypeptide.
 2. Theimmunoassay of claim 1 in which said HIV-1 mutein is strain SF2.
 3. Theimmunoassay of claim 1 in which at least hypervariable region V₁ isdeleted from said mutein.
 4. The immunoassay of claim 1 in which atleast hypervariable region V₂ is deleted from said mutein.
 5. Theimmunoassay of claim 1 in which at least hypervariable region V₃ isdeleted from said mutein.
 6. The immunoassay of claim 1 in which atleast hypervariable region V₄ is deleted from said mutein.
 7. Theimmunoassay of claim 1 in which at least hypervariable region V₅ isdeleted from said mutein.
 8. The immunoassay of claim 1 in which V₁ isdeleted from said mutein.
 9. The immunoassay of claim 1 in which V₂ isdeleted from said mutein.
 10. The immunoassay of claim 1 in which V₃ isdeleted from said mutein.
 11. The immunoassay of claim 1 in which V₄ isdeleted from said mutein.
 12. The immunoassay of claim 1 in which V₅ isdeleted from said mutein.
 13. The immunoassay of claim 1 in which V₁ andV₂ are deleted from said mutein.
 14. The immunoassay of claim 1 in whichV₃, V₄ and V₅ are deleted from said mutein.
 15. The immunoassay of claim1 in which V₁ through V₅ are deleted from said mutein.
 16. Theimmunoassay of claim 1 in which the liquid sample is serum.
 17. Theimmunoassay of claim 16 in which said serum is human serum.